Artificial nucleic acid molecules for improved protein expression
The invention relates to an artificial nucleic acid molecule comprising an open reading frame and a 3′-UTR comprising at least one poly(A) sequence or a polyadenylation signal. The invention further relates to a vector comprising the artificial nucleic acid molecule comprising an open reading frame and a 3′-UTR comprising at least one poly(A) sequence or a polyadenylation signal, to a cell comprising the artificial nucleic acid molecule or the vector, to a pharmaceutical composition comprising the artificial nucleic acid molecule or the vector and to a kit comprising the artificial nucleic acid molecule, the vector and/or the pharmaceutical composition. The invention also relates to a method for increasing protein production from an artificial nucleic acid molecule and to the use of a 3′-UTR for a method for increasing protein production from an artificial nucleic acid molecule. Moreover, the invention concerns the use of the artificial nucleic acid molecule, the vector, the kit or the pharmaceutical composition as a medicament, as a vaccine or in gene therapy.
1. A method for increasing protein production from a RNA molecule comprising providing the RNA molecule comprising:
a) a 5′-cap structure;
b) at least one open reading frame (ORF) encoding a protein; and
c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
(i) the first poly(A) sequence comprises at least 20 adenine nucleotides; and
(ii) the second poly(A) sequence comprises at least 70 adenine nucleotides,
wherein the first and the second poly(A) sequences are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides and having no more than 2 consecutive adenine nucleotides,
wherein the RNA molecule yields increased protein production when expressed in a cell or an organism in comparison to a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking a second poly(A) sequence.
2. The method of claim 1 , wherein at least one of the poly(A) sequences is located at the 3′ terminus of the RNA molecule.
3. The method of claim 1 , wherein the RNA molecule comprises a heterologous 5′ UTR sequence.
4. The method of claim 1 , wherein the protein is an antigen.
5. The method of claim 4 , wherein the antigen is an antigen from a bacterial, a viral, a fungal or a protozoan pathogen.
6. The method of claim 4 , wherein the antigen is a viral antigen.
7. The method of claim 4 , wherein the antigen is a tumor antigen.
8. The method of claim 1 , wherein the RNA molecule comprises from 5′ to 3′:
I) the 5′-cap structure;
II) the heterologous 5′ UTR sequence;
III) the at least one open reading frame (ORF) encoding a protein;
IV) the heterologous 3′-untranslated region (3′-UTR) comprising, from 5′ to 3′,
(i) the first poly(A) sequence comprising at least 20 adenine nucleotides;
(ii) the nucleic acid sequence which separates the first and the second poly(A) sequence, comprising from 10 to 90 nucleotides and having no more than 2 consecutive adenine nucleotides; and
(iii) the second poly(A) sequence comprising at least 70 adenine nucleotides.
9. The method of claim 8 , wherein the ORF encoding the protein has a G/C content that is increased relative to a corresponding reference ORF encoding the protein.
10. The method of claim 9 , wherein the ORF encoding the protein has a G/C content that is increased by at least 7% relative to a corresponding reference ORF encoding the protein.
11. The method of claim 10 , wherein the ORF encoding the protein has a G/C content that is increased by at least 15% relative to a corresponding reference ORF encoding the protein.
12. The method of claim 11 , wherein the first poly(A) sequence comprises at least 30 adenine nucleotides.
13. The method of claim 12 , wherein the nucleotide sequence which separates the first and the second poly(A) sequences consists of 10 nucleotides and has no more than 2 consecutive adenine nucleotides.
14. The method of claim 13 , wherein the RNA molecule comprises at least one nucleotide analog.
15. The method of claim 14 , wherein the at least one nucleotide analogue is a modified form of uridine.
16. The method of claim 15 , wherein the modified form of uridine is chemically altered by methylation.
17. The method of claim 16 , wherein the modified form of uridine is a naturally occurring variant of uridine.
18. The method of claim 17 , wherein the RNA molecule is complexed with a cationic or polycationic compound.
19. The method of claim 18 , wherein the cationic or polycationic compound comprises a cationic or polycationic peptide.
20. The method of claim 18 , wherein the cationic or polycationic compound comprises a cationic lipid.
21. A method for increasing protein production from a RNA molecule comprising providing the RNA molecule formulated in a pharmaceutical composition, where the RNA molecule comprises in the 5′ to 3′ direction:
a) a 5′-cap structure;
b) at least one open reading frame (ORF) encoding a protein that is a viral antigen; and
c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
(i) the first poly(A) sequence comprises at least 20 adenine nucleotides; and
(ii) the second poly(A) sequence comprises at least 70 adenine nucleotides,
wherein the first and the second poly(A) sequences are separated by a nucleic acid sequence comprising from 10 to 90 nucleotides and having no more than 2 consecutive adenine nucleotides,
wherein the RNA molecule comprises at least one nucleotide analogue, which is a naturally occurring variant of uridine,
wherein the RNA molecule yields increased protein production when expressed in a cell or an organism in comparison to a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking a second poly(A) sequence;
wherein the ORF encoding the viral antigen has a G/C content that is increased by at least 15% relative to a corresponding reference ORF; and
wherein the RNA molecule is complexed with a cationic or polycationic compound comprising a cationic lipid.
22. The method of claim 21 , wherein the naturally occurring variant of uridine is chemically altered by methylation.
23. The method of claim 21 , wherein the RNA molecule is complexed with a cationic carrier or a polycationic carrier.
24. The method of claim 23 , wherein the cationic or polycationic compound comprises a cationic lipid.
25. A method for increasing protein production from a RNA molecule comprising providing the RNA molecule formulated in a pharmaceutical composition, where the RNA molecule comprises:
a) a 5′-cap structure;
b) at least one open reading frame (ORF) encoding a protein; and
c) a heterologous 3′-untranslated region (3′-UTR) comprising at least a first and a second poly(A) sequence, wherein:
(i) the first poly(A) sequence comprises at least 20 adenine nucleotides; and
(ii) the second poly(A) sequence comprises at least 70 adenine nucleotides,
wherein the first and the second poly(A) sequences are separated by a nucleic acid sequence consisting of 10 nucleotides and having no more than 2 consecutive adenine nucleotides,
wherein the ORF encoding the protein has a G/C content that is increased by at least 15% relative to a corresponding reference ORF encoding the protein,
wherein the RNA molecule yields increased protein production when expressed in a cell or an organism in comparison to a reference nucleic acid molecule comprising an identical nucleic acid sequence as the RNA molecule but lacking a second poly(A) sequence.
26. The method of claim 25 , wherein the ORF encoding the protein is an antigen.
27. The method of claim 26 , wherein the antigen is a viral antigen.
28. The method of claim 27 , wherein the RNA molecule comprises at least one nucleotide analogue, which is a naturally occurring variant of uridine.
29. The method of claim 28 , wherein the RNA molecule is complexed with a cationic carrier or a polycationic carrier.
30. The method of claim 29 , wherein the cationic or polycationic compound comprises a cationic lipid.